Nociceptor Neurons Magnify Host Responses to Aggravate Periodontitis

Abstract

Periodontitis is a highly prevalent chronic inflammatory disease that progressively destroys the structures supporting teeth, leading to tooth loss. Periodontal tissue is innervated by abundant pain-sensing primary afferents expressing neuropeptides and transient receptor potential vanilloid 1 (TRPV1). However, the roles of nociceptive nerves in periodontitis and bone destruction are controversial. The placement of ligature around the maxillary second molar or the oral inoculation of pathogenic bacteria induced alveolar bone destruction in mice. Chemical ablation of nociceptive neurons in the trigeminal ganglia achieved by intraganglionic injection of resiniferatoxin decreased bone loss in mouse models of experimental periodontitis. Consistently, ablation of nociceptive neurons decreased the number of osteoclasts in alveolar bone under periodontitis. The roles of nociceptors were also determined by the functional inhibition of TRPV1-expressing trigeminal afferents using an inhibitory designer receptor exclusively activated by designer drugs (DREADD) receptor. Noninvasive chemogenetic functional silencing of TRPV1-expressing trigeminal afferents not only decreased induction but also reduced the progression of bone loss in periodontitis. The infiltration of leukocytes and neutrophils to the periodontium increased at the site of ligature, which was accompanied by increased amount of proinflammatory cytokines, such as receptor activator of nuclear factor κΒ ligand, tumor necrosis factor, and interleukin 1β. The extents of increase in immune cell infiltration and cytokines were significantly lower in mice with nociceptor ablation. In contrast, the ablation of nociceptors did not alter the periodontal microbiome under the conditions of control and periodontitis. Altogether, these results indicate that TRPV1-expressing afferents increase bone destruction in periodontitis by promoting hyperactive host responses in the periodontium. We suggest that specific targeting of neuroimmune and neuroskeletal regulation can offer promising therapeutic targets for periodontitis supplementing conventional treatments.

Keywords: TRPV1; bone resorption; chemogenetics; nociceptors; primary afferents; resiniferatoxin.

Figure 1.

Ablation of transient receptor potential vanilloid 1⁺ (TRPV1⁺) trigeminal afferents attenuated periodontitis-induced bone loss in mice. (A) Timeline of an experiment into ligature-induced periodontitis. (Bottom left) For selective ablation of TRPV1⁺ afferents, resiniferatoxin (RTX, 50 ng in 0.5 µL PBS) was stereotaxically microinjected into the bilateral trigeminal ganglia (TG) in adult C57BL/6 mice. The vehicle (Veh)–injected group served as control. (Bottom right) After a week, a ligature (5–0 silk, blue arrowhead) was tied around the maxillary left second molar (Mx Lt M2), and an unligatured contralateral tooth served as a control. After 2 wk, the mice were euthanized for micro–focus computed tomography (µCT) and histology study. Scale bar: 1 mm. (B) Three-dimensional (3D) reconstruction of µCT scanned images. ABC, alveolar bone crest; CEJ, cementoenamel junction; the red arrows indicate points where distances between the ABC and the CEJ were evaluated. Four measurements were averaged in each sample. Scale bar: 1 mm. (C) Distances from the ABC to the CEJ assessed in µCT. ***P < 0.001 in Bonferroni post hoc tests following 2-way analysis of variance. n = 6 to 9 per group. (D) Timeline of an experiment for bacteria-induced periodontitis. In C57BL/6 mice, antibiotics were administered through drinking water for 8 d, followed by 2 d of normal drinking water. Oral inoculation of Porphyromonas gingivalis (2 × 10⁹ colony-forming units [CFU], 200 µL) and Fusobacterium nucleatum (2 × 10⁹ CFU, 200 µL) or vehicle (2% methylcellulose) only was performed over 12 d (6 times every 2 d). Mice were euthanized 2 wk after the last inoculation. (E) Examples of 3D reconstructed µCT images. Nine measurements were averaged in each sample. Scale bar: 1 mm. (F) RTX-induced ablation of TRPV1⁺ afferents reduced bacterial inoculation-induced resorption of alveolar bone. Averaged distances from the ABC to the CEJ assessed in µCT. *P < 0.05 in Bonferroni post hoc tests following 2-way analysis of variance. n = 4 in the control and 5 in the bacteria group.

Figure 2.

Ablation of transient receptor potential vanilloid 1⁺ (TRPV1⁺) trigeminal afferents attenuated osteoclasts in alveolar bone under periodontitis without altering circulating norepinephrine. (A) Histological evaluation of osteoclasts by tartrate-resistant acid phosphatase (TRAP) staining. Arrowheads indicate TRAP-positive cells. Scale bar: 100 µm. (B) Number of osteoclasts. Multinucleated dark red cells lying along the alveolar bone surface around M2 were counted. ****P < 0.0001 in Bonferroni post hoc tests following 2-way analysis of variance. n = 6 per group. (C) The effects of intra–trigeminal ganglia (TG) injection of resiniferatoxin (RTX) on wiping behaviors in response to ocular application of capsaicin from a cohort of mice used in Fig. 1A. n = 6 or 8 per group. (D) Ablation of TRPV1⁺ neurons in TG after RTX injection. (E) Enzyme-linked immunosorbent assay for evaluating circulating levels of norepinephrine in blood collected 3 wk after TG injection of RTX or vehicle. n = 6 or 8 per group.

Figure 3.

Noninvasive chemogenetic inhibition of transient receptor potential vanilloid 1⁺ (TRPV1⁺) afferents reduces ligature-induced bone loss. (A) Projection of TRPV1-lineage afferent terminals in interproximal gingiva. TRPV1^cre;Rosa26^mTmG mice express membrane-bound green fluorescent protein (GFP) in TRPV1-lineage nerve terminals (left) and tdTomato (middle) in all other cells. Blue in merged image, DAPI. CB, crestal bone. Scale bar: 100 µm. (B–D) Experimental protocol (B). A ligature was placed in Trpv1^Cre mice (Cre) or TRPV1^Cre;R26^LSL-hM4Di mice (hM4Di). To activate hM4Di, an inhibitory designer receptor exclusively activated by designer drugs (DREADD) receptor, an osmotic pump (OP) was implanted under the back skin to continuously release clozapine-N-oxide (CNO; 0.25 mL/h for 2 wk). After 2 wk, the mice were euthanized for micro–focus computed tomography (µCT) assay. Examples of µCT images are shown (C). Scale bar: 0.5 mm. Bone loss evaluated in µCT (D). *P < 0.05 in unpaired Student’s t tests. n = 3 or 4 per group. (E) Experimental protocol. In Trpv1^Cre mice, adeno-associated virus (AAV) encoding cre-dependent hM4Di fused with a fluorescent reporter (mCherry) under a neuronal promoter (human synapsin) (AAV5-hSyn-DIO-HA-hM4D(Gi)-mCherry; abbreviated to AAV-DIO-hM4Di) or GFP (AAV5-DIO-GFP) was microinjected into the left trigeminal ganglia (TG) (0.5 µL). After 3 wk, the ligature was placed on Mx Lt M2. X marks indicate the time points of euthanasia. In group a, AAV-DIO-GFP was injected without CNO administration. In protocols b and c, AAV-DIO-hM4Di or AAV-DIO-GFP was injected, and the OP was implanted 1 wk after ligature when CNO administration began. CNO was administered for the following 2 wk, and then the mice were euthanized. (F) Bone loss evaluated in µCT. **P < 0.005 in Sidak post hoc tests following 1-way analysis of variance. n = 7 to 8 per group. (G) Labeling of GFP and TRPV1 in AAV-DIO-GFP-injected TG. Scale bar: 50 µm.

Figure 4.

Ablation of transient receptor potential vanilloid 1⁺ (TRPV1⁺) afferents decreases host responses in the periodontium under periodontitis. (A, B) Flow cytometry was performed to identify the proportion of immune cells in single-cell suspensions from gingiva in control or ligature side 2 wk after placing the ligature in mice with intra–trigeminal ganglia (TG) injection of vehicle (V) or resiniferatoxin (RTX) (R). The percentage in each plot represents the fraction of the given cells out of live single cells. Examples of CD45⁺ leukocytes (A) and Ly6G⁺ CD11b⁺ neutrophils (B) in vehicle (Veh)/control (a), RTX/control (b), Veh/ligature (c), and RTX/ligature (d) groups. Proportions of immune cells in live single cells in each sample are plotted (e). ****P < 0.0001 in Sidak post hoc tests following 1-way analysis of variance. n = 6 in all groups. (C, D) Luminex assay for measuring cytokines (C) or real-time polymerase chain reaction assay for evaluating gene expression (D) in periodontium from control (Con) or ligature (Lig) side after intra-TG injection of Veh or RTX (RTX). The mice were euthanized 2 wk after placing the ligature. **P < 0.005 and ***P < 0.0005 in Sidak post hoc tests following 1-way analysis of variance. n = 10 (C) or 9 per group (D).

Figure 5.

Ablation of transient receptor potential vanilloid 1⁺ (TRPV1⁺) afferents does not alter microbial dysbiosis under periodontitis. (A) Time course of the experiment to evaluate the periodontal microbiome. One week after intra–trigeminal ganglia (TG) injection of vehicle (V) or resiniferatoxin (R), ligatures were placed and then recovered either after 3 h (control) or 2 wk (periodontitis). Microbial genomic DNA was isolated from the ligatures, and bacterial 16S ribosomal RNA (rRNA) sequencing was performed. (B) Taxonomic composition of each sample at the phylum level. (C) Richness and evenness in the samples were evaluated using the Shannon diversity index. n = 5 (V/3h), 6 (R/3h), 9 (V/2w), and 9 (R/2w). P = 0.58 in one-way analysis of variance. (D) Principal coordinates analysis (PCoA) plot was generated from Brady–Curtis dissimilarity data. P = 0.007 in permutational multivariate analysis of variance. Post hoc pairwise comparisons; P = 0.85 between Veh/3h versus RTX/3h, P = 0.02 between Veh/3h versus Veh/2w, P = 0.02 between Veh/3h versus RTX/2w, P = 0.03 between RTX/3h versus Veh/2w, P = 0.02 between RTX/3h versus RTX/2w, and P = 0.85 between Veh/2w versus RTX/2w. (E) Four examples of differentially abundant taxa among group variables. Differential abundance testing (DESeq2, R package) identified 15 operational taxonomic units that were differentially abundant among treatments, 4 of which are shown. 2w, 2 wk after ligature placement; 3h, 3 h after ligature placement; FDR, false discovery rate; RTX, resiniferatoxin injection into TG; Veh, vehicle injection into TG. n = 5 (Veh/3h), 6 (RTX/3h), 9 (Veh/2w), and 9 (RTX/2w).